In the last year, we have continued our studies that investigate how cells divide and differentiate in an effort to understand how these processes may fail during disease. Specifically, this report will outline progress that we have made in the past year that extend our studies on how proteins localize and assemble during growth and development of the model organism Bacillus subtilis. First, we have investigated the irreversible assembly of a novel cytoskeletal protein called SpoIVA that, unlike proteins which form dynamic structures, uses ATP hydrolysis to drive its assembly (not disassembly). Second, we reported the identification of a previously un-annotated gene in B. subtilis (that we have named cmpA) that participates in a previously unknown pathway that orchestrates the morphogenesis of two large structures during spore formation. Third, we reported that a cell division protein called DivIVA exploits changes in cell shape to spatially and temporally ensure the faithful elaboration of a single division septum at mid-cell during growth. Mature spores of B. subtilis are encased in a proteinaceous shell called the coat that is composed of about seventy different proteins. These proteins assemble atop a platform created by a single structural protein called SpoIVA. Our previous studies had shown that SpoIVA is an ATPase, which we thought was an unusual activity for a static morphogenetic protein, and proposed that ATP hydrolysis may drive the assembly into a stable structure surrounding spores. To begin to rigorously test this hypothesis, we initiated a multidisciplinary collaboration with the bioinformatics group of L. Aravind in NCBI, who were able to construct a putative topology diagram of the protein and predict key residues that were specifically required for ATP hydrolysis, and not ATP binding. We then established two in vitro assays, including a biophysical assay that was done in collaboration with a postdoctoral fellow in James Sellers? group in NHLBI, to measure the assembly of wild type SpoIVA and several variants that were deficient in ATP hydrolysis. Our results indicated that SpoIVA assembles into a static structure that does not readily disassemble; that this assembly requires ATP hydrolysis, not simply ATP binding; and that ATP hydrolysis results in a structural change in SpoIVA that drives polymerization into a largely nucleotide-free polymer. Moreover, bioinformatic analysis of SpoIVA revealed that the protein is a newly identified member of the TRAFAC class of GTPases (which includes signaling proteins like Ras, membrane remodeling proteins like dynamin, and motor proteins like myosin and kinesin) and that SpoIVA likely evolved as a result of an ancient gene duplication event of a highly conserved GTP-binding translation factor, followed by rapid divergent evolution. This divergence included the loss of GTP binding (and subsequent acquiring of ATP-binding specificity) and the addition of both a C-terminal polymerization domain and a membrane anchor domain. We proposed that nucleotide hydrolysis-dependent polymerization of static polymers may be a conserved mechanism by which durable biological structures may be built. The results of this work have been submitted for publication. SpoIVA is anchored onto the surface of the developing spore by a small amphipathic helical protein called SpoVM. Previously, we reported that the proper subcellular localization of this small protein is dictated by a geometric cue: in this case, the convex membrane curvature present only on the surface of the developing spore. Spores are encased in a second shell, made of peptidoglycan, which lies beneath the coat and is called the cortex. For over four decades, it has been known that cortex assembly only initiates after proper initiation of coat assembly, but the mechanisms that mediate this orchestrated assembly were not known. In order to understand this, we first identified mutant alleles of spoVMthat were specifically defective in assembling the cortex, but not the coat. We then identified a suppressor mutation that corrected this defect, and discovered that this mutation mapped to a previously un-annotated 37-codon-long open reading frame that we have named cortex morphogenetic protein A (cmpA). Deletion of this gene resulted in premature cortex assembly, whereas overexpression of cmpA resulted in repression of cortex assembly. We proposed that CmpA is a checkpoint that represses cortex assembly until the proper time (successful initiation of coat assembly) whereupon the repressive activity of CmpA is relieved post-translationally. These results were published in May, 2012 and current efforts in the lab are oriented towards identifying the downstream target of CmpA inhibition, understanding the mechanism by which CmpA inhibition is removed, and identifying other genes that participate in this pathway. During cell division in B. subtilis, a group of proteins that comprise the Min system assembles at mid-cell in order to ensure that only a single septum forms. Disrupting this quality control mechanism results in the formation of minicells: spherical cells that are devoid of genetic material. However, it had been unclear how 1) the Min system localized to mid-cell at the correct time, and 2) what prevented the Min system from inhibiting proper cell division at mid-cell, while preventing cell division on either side. Recently, we proposed that the cell division protein DivIVA, which recruits the Min system, preferentially localized to regions displaying high concave membrane curvature. Consistent with this notion, we found that DivIVA-GFP using deconvolution microscopy and discovered that DivIVA localized to mid-cell only after the onset of membrane invagination and that DivIVA did not localize to division sites where the division machinery had simply assembled but was not functional. By examining DivIVA stability using FRAP experiments, we observed that, unlike other components of the division machinery, DivIVA localized as a stable, not dynamic, ring at mid-cell that was composed largely of newly synthesized protein. Furthermore, in collaboration with Joe Pogliano?s group at U.C. San Diego, we employed a super-resolution microscopy technique to demonstrate that DivIVA actually assembles into two adjacent rings that flank the division septum at mid-cell on either side. Taken together, we have proposed that DivIVA temporally regulates the Min system by arriving at the division only after the onset of membrane constriction; and spatially regulates the Min system by forming a barrier (two separated rings) that physically keeps it separated from the constricting division machinery at mid-cell. These results were published in December, 2011, and we are currently examining the kinetics of DivIVA assembly at the single molecule level in collaboration with Hari Shroff?s group in NIBIB.